Cell populations enriched for stem cells are often desired for use in research or therapy. Typical sources of stem cells include bone marrow, whole peripheral blood, leukopheresis or apheresis products, especially from “mobilized” donors, or other less common sources, such as umbilical cord blood and tissue or organ suspensions. Enrichment of stem cells has been done in several ways. Typical methods include density step gradients (e.g., FICOLL-HYPAQUE®, colloidal silica, and the like), elutriation, centrifugation, lysis of erythrocytes by hypotonic shock, and various combinations of these methods. As an example, the purification of stem cells from bone marrow requires removal of erythrocytes and granulocytes, which is often accomplished by FICOLL-HYPAQUE® density gradient centrifugation. There are disadvantages to each of these methods, one of which is the need for laborious washing steps after the enrichment step is performed, e.g., to remove the density gradient centrifugation medium.
Following enrichment, the cells are typically washed by a repetitive process. The steps generally include placing the enriched cell suspension into a centrifuge tube and pelleting the cells to the bottom of the tube by use of a centrifuge. The tube is removed from the centrifuge, and the supernatant is decanted from the pelleted cells. A wash liquid is added to the tube, and the cell pellet is resuspended. These steps are typically repeated 2 to 4 times.
One disadvantage of this washing process is that sequential resuspension and centrifugation can decrease cell viability and increase cell lysis. Another disadvantage of washing by centrifugation is the opportunity for bacteria or other infectious agents to contaminate the cells. Even if all the materials are kept sterile, the repeated opening of the centrifuge tubes, and the exposure of pipettes and bottles of wash solution to the air can result in contamination. The risk of contamination is sufficiently significant that some medical regulatory agencies have demanded that only “closed” systems are used for cell handling.
Filtration methods have also been used to remove cells from blood while retaining other blood constituents for later use. Such methods generally trap the cells on a filter in a non-recoverable form, while allowing other blood constituents to pass through the filter and into a collection vessel. For example, filters are available to remove leukocytes from blood so that the incidence of alloimmune reactions is minimized following blood transfusions. Leukocyte removal is typically done using filters which are made of matted plastic fiber mesh. The mesh is usually arranged to trap the leukocytes in a reticulated matrix having enough depth so that the cells are trapped throughout the depth of the filter, thereby keeping the filter from clogging, as would occur if the leukocytes were trapped on a planar surface.
In addition to the physical trapping of the cells, the materials and large surface area of the filter allow leukocytes to adhere irreversibly to the surface. Many of these adherent cells are the very ones desired for some medical procedures. The resulting combination of trapping and adherence to the filter creates a highly efficient means of removing the leukocytes for disposal prior to blood infusion therapy. However, when leukocytes are the desired cells, this method of filtration is not advantageous.
A method that has been useful in the fractionation of various particles is tangential flow filtration (TFF) or “cross-flow” filtration. TFF relies on the movement of a fluid parallel to the surface of a porous membrane filter. The pores of the membrane allow passage of the fluid and of particles within the fluid that are typically smaller than the pores. In addition, the cross-flow (or “tangential” flow) of fluid parallel to the filter prevents a build-up of particles larger than the pores on the filter surface.
TFF has been used for the gross separation of various materials. The use of tangential flow filtration in the pharmaceutical field has been reviewed by Genovesi (J. Parenter. Aci. Technol., 37:81, 1983), including the filtration of sterile water for injection, clarification of a solvent system, and filtration of enzymes from broths and bacterial cultures. Marinaccio et al. (WO 85/03011) report a process for use in the removal of particulate blood components from blood for plasmapheresis, and Robinson et al. (U.S. Pat. No. 5,423,738) describe the use of TFF for the removal of plasma from blood, allowing the reinfusion of blood cells and platelets into patients.
In another use, TFF has been reported for the filtration of beer (EP 0 208 450), specifically for the removal of particulates such as yeast cells and other suspended solids. Kothe et al. (U.S. Pat. No. 4,644,056) disclose the use of TFF in the purification of immunoglobulins from milk or colostrurn, and Castino (U.S. Pat. No. 4,420,398) describes its use in the separation of antiviral substances, such as interferons, from broths containing these substances as well as viral particles and cells. Similarly, TFF has been used in the separation of bacterial enzymes from cell debris. (Quirk et al., Enzyme Microb. Technol., 6:201, 1984). In addition, tangential flow filtration units have been employed in the concentration of cells suspended in culture media. (See, e.g., Radlett, J. Appl. Chem. Biotechnol., 22:495, 1972).
TFF has also been reported to separate liposomes and lipid particles according to size. (Lenk et al., U.S. Pat. No. 5,948,441). TFF allows for the formation and isolation of liposomes and lipid particles having a defined size range from heterogeneous populations of such particles. (See Lenk et al., supra).
However, while TFF has been used for gross fractionation of biological liquids and the separation of, for example, liposomes, the use of TFF for separation of live cell populations differing in defined characteristics has not been appreciated in the art. In particular, the unique problems associated with the selective separation of stem cells from other bone marrow cells or from blood cells and tissue or organ suspensions while maintaining sterility, cell viability and regenerative activity has not been addressed. In addition, the removal of other cell populations such as, e.g., populations with overlapping size ranges, has not been solved by current approaches.
Therefore, there remains a need in the art for additional devices and methods for selectively enriching stem cells from other bone marrow or blood constituents, including plasma, erythrocytes, and/or platelets, while preserving sterility, cell viability and regenerative and cellular activity. The present invention satisfies these and other needs.